Process for the preparation of qs21 intermediates
The QS21 intermediate was synthesized by in vitro enzymatic catalysis using E1-CSLM2 enzyme expressed in insect sf9 and E2-GmSGT2 enzyme expressed in Escherichia coli, which simplified the synthesis steps and improved the yield.
Patent Information
- Application Number
- CN202511447767.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2025-12-26
- Estimated Expiration
- 2045-10-11
AI Technical Summary
Existing methods for synthesizing QS21 are complex and have low extraction rates.
The QA-GlcA intermediate was synthesized via an in vitro enzymatic method using E1-CSLM2 enzyme catalysis, followed by E2-GmSGT2 enzyme catalysis to obtain the QS21 intermediate QA-GlcA-Gal. The catalytic efficiency was improved by expressing E1-CSLM2 enzyme in insect sf9 and E2-GmSGT2 enzyme in Escherichia coli.
A simplified synthesis of the QS21 intermediate was achieved, improving the yield.
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Figure CN120905346B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a preparation method of a QS21 intermediate. BACKGROUND
[0002] The QS21 intermediate is a derivative of quillic acid, and these compounds are the core components of the structure of QS21. QS21 belongs to triterpene saponins, and the molecular structure comprises four key regions: a lipophilic triterpene core as a main skeleton, a branched trisaccharide at the C3 position, a linear tetrasaccharide chain at the C28 position, and an acyl side chain connected by an ester bond. At present, the methods for synthesizing QS21 include chemical synthesis, plant cell culture and extraction, but these synthesis methods are complex in steps and low in extraction rate. SUMMARY
[0003] In view of this, the present application provides a preparation method of a QS21 intermediate, which adopts an in vitro enzyme method, synthesizes a QA-GlcA intermediate through E1-CSLM2 enzyme catalysis, and obtains the QS21 intermediate (QA-GlcA-Gal) through E2-GmSGT2 enzyme catalysis of the QA-GlcA intermediate, which is simple in steps and high in yield.
[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0005] The present application provides a preparation method of a QS21 intermediate, comprising the following steps:
[0006] S1, purifying a QA-GlcA intermediate after mixing and reacting QA quillaja acid, UDP-D-GlcA and E1-CSLM2 enzyme;
[0007] S2, purifying a QS21 intermediate QA-GlcA-Gal after mixing and reacting the QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme.
[0008] In some embodiments, the enzyme catalysis reaction of step S1 is as follows:
[0009]
[0010] In some embodiments, the enzyme catalysis reaction of step S2 is as follows:
[0011]
[0012] Preferably, in step S1, the E1-CSLM2 enzyme is obtained by insect sf9 expression.
[0013] Preferably, in step S1, the mixed reaction substances further comprise: pH 7.5 Tris-HCl, MgCl2 and DMSO.
[0014] Preferably, in step S1, after mixing, the final concentration of QA Quillaja Saponins is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.
[0015] Preferably, in step S1, the temperature of the mixed reaction is 30℃.
[0016] Preferably, in step S2, the E2-GmSGT2 enzyme is obtained by expression of E. coli.
[0017] Preferably, in step S2, the mixed reaction substances further comprise: pH 7.5 Tris-HCl and MgCl2.
[0018] Preferably, in step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.
[0019] Preferably, the temperature of the mixed reaction is 30℃.
[0020] Compared with the prior art, the present application has the following beneficial effects:
[0021] (1) The present application catalyzes the reaction of QA Quillaja Saponins by E1-CSLM2 enzyme to obtain QA-GlcA intermediate, and then catalyzes the reaction of QA-GlcA intermediate by E2-GmSGT2 enzyme to obtain intermediate QS21-GlcA-Gal. The steps are simple and the yield is high.
[0022] (2) In order to improve the catalytic efficiency of E1-CSLM2 enzyme and E2-GmSGT2 enzyme, the present application uses insect sf9 to express E1-CSLM2 enzyme and uses E. coli to express E2-GmSGT2. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Embodiment 3 of the present application (sf9 expression); E. coli The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Embodiment 3 of the present application (sf9 expression);
[0024] Figure 2 The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Embodiment 3 of the present application (sf9 expression);
[0025] Figure 3The HPLC detection chart of the intermediate product QA-GlcA provided in Embodiment 3 of the present application;
[0026] Figure 4 The activity detection mass spectrum chart of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application; E. coli The activity detection mass spectrum chart of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application;
[0027] Figure 5 The activity detection mass spectrum chart of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application;
[0028] Figure 6 The activity detection mass spectrum chart of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application; E. coli The activity detection mass spectrum chart of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application;
[0029] Figure 7 The HPLC detection chart of the intermediate product QA-GlcA-Gal provided in Embodiment 3 of the present application;
[0030] Figure 8 The nuclear magnetic detection chart of the intermediate product QA-GlcA-Gal provided in Embodiment 3 of the present application. DETAILED DESCRIPTION
[0031] The present application will be further described in conjunction with specific embodiments so that those skilled in the art can more clearly understand the present application.
[0032] QS21 intermediates are derivatives of quillic acid, which are the core components of QS21 structure. QS21 belongs to triterpene saponins, and its molecular structure contains four key regions: a lipophilic triterpene core as the main skeleton, a branched trisaccharide at the C3 position, a linear tetrasaccharide chain at the C28 position, and an acyl side chain connected by an ester bond. Currently, the methods for synthesizing QS21 include chemical synthesis, plant cell culture and extraction, but these synthesis methods are complex and have low extraction rates.
[0033] To solve the above technical problems, the present application provides a preparation method of QS21 intermediate, comprising the following steps:
[0034] S1, purifying the mixture of QA quillic acid, UDP-D-GlcA and E1-CSLM2 enzyme after reaction to obtain QA-GlcA intermediate;
[0035] S2, purifying the mixture of QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme after reaction to obtain QS21 intermediate QA-GlcA-Gal.
[0036] Further, in step S1, the E1-CSLM2 enzyme is obtained by insect sf9 expression.
[0037] Further, in step S1, the mixed reaction substances further include: pH 7.5 Tris-HCl, MgCl2, and DMSO.
[0038] Further, in step S1, after mixing, the final concentration of QA quillaic acid is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.
[0039] Further, in step S1, the temperature of the mixed reaction is 30°C.
[0040] Further, in step S2, the E2-GmSGT2 enzyme is obtained by E. coli expression.
[0041] Further, in step S2, the mixed reaction substances further include: pH 7.5 Tris-HCl and MgCl2.
[0042] Further, in step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.
[0043] Further, the temperature of the mixed reaction is 30°C.
[0044] Example 1 Insect cell expression of E1-CSLM2 and E2-UGT73CU3
[0045] After the target gene (CSLM2, UGT73CU3) is synthesized, it is cloned into a baculovirus transfer vector (such as pFastBac1) and transformed into DH10Bac competent cells. A white single colony is inoculated in 5 mL of LB medium containing 50 μg / mL of kanamycin and incubated at 37°C for 16 hours. Recombinant Bacmid DNA is extracted using an alkaline lysis method. 5 μg of recombinant Bacmid and 10 μL of Cellfectin II reagent are diluted in 100 μL of antibiotic-free SFM, mixed, and incubated at room temperature for 20 minutes. Discard the cell culture medium, add the DNA-liposome complex, incubate at 27°C for 5 hours, replace the fresh culture medium, and continue to culture for 96 hours to harvest the P1 virus supernatant. The P1 virus is inoculated into sf9 cells (density 2 x 10 6Cells / mL), cultured at 27℃ for 96 hours. Take the cell culture medium, incubate at 4℃ and 7000 rpm for 10 min, collect the precipitate, resuspend the precipitate with PBS, and sonicate (15% 3s / 3s 10min), collect the crude enzyme disruption solution for later use.
[0046] Example 2: Expression of E1-CSLM2, E2-UGT73CU3, and E2-GmSGT2 in Escherichia coli
[0047] Will come from Quillaja saponaria The CSLM2 gene, UGT73CU3 gene, and GmSGT2 gene derived from soybean were cloned into pMAL-c2X, transformed into 100 μL of BL21(DE3) competent cells, and plated on ampicillin plates for overnight culture. Single colonies were picked and transferred to 10 mL of medium containing 100 μg / mL ampicillin, cultured at 37°C for 6 hours, and then transferred at a 1% (v / v) ratio to 400 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37°C until OD500. 600 =0.7, add 0.2mM IPTG to a final concentration, incubate overnight at 20℃, and collect bacterial cells by centrifugation at 5000g. Resuspend the bacterial cells in 10 volumes of 20mM Tris-HCl (pH 7.5), 500mM NaCl, and 0.1% Triton X-100 solution, and disrupt the cells using an ultrasonic cell disruptor. After centrifugation at 12000g, aspirate the supernatant, filter through a 0.45μm filter membrane, and purify the fusion protein in one step using an Amylose resin column.
[0048] Example 3 Synthesis of QS21 intermediate QA-GlcA-Gal
[0049] 1. Catalytic synthesis of QA-GlcA intermediate
[0050] The catalytic reaction system is shown in Table 1.
[0051] Table 1
[0052]
[0053] The enzyme activity was detected using the reaction system shown in Table 1, and the purity of the catalytically obtained product was also tested. The results are shown in the table below. Figures 1-3 .
[0054] from Figure 1 It can be seen that, E. coli The E1-CSLM2 protein expressed in *E. coli* showed no activity (substrate molecular weight 661 was not detected) as the reaction did not occur according to mass spectrometry. Figure 2It can be seen that the SF9 expressed E1-CSLM2 protein, mass spectrometry results show that the reaction is normal (detecting the substrate molecular weight 661).
[0055] Therefore, the SF9 expressed E1-CSLM2 protein is amplified by the system in Table 1, the obtained product is purified by liquid phase preparation, and the purity of the product is detected by HPLC after freeze-drying. Figure 3 It can be seen that the purity of the product detected by HPLC is 99% (excluding solvent peaks).
[0056] 2. Catalytic synthesis of QA-GlcA-Gal intermediate
[0057] The catalytic reaction system is shown in Table 2.
[0058] Table 2
[0059]
[0060] According to the reaction system in Table 2, the enzyme activity is detected, and the detection results are shown in Table 3. Figures 4-6 .
[0061] From the detection results of the SF9 expressed E2-GmSGT2 protein, it can be seen that the reaction is normal (detecting the product molecular weight 823, and not detecting the substrate molecular weight 661); the SF9 expressed E2-UGT73CU3 protein, mass spectrometry detects that the reaction is normal, but the reaction efficiency is low (detecting the product and substrate molecular weight at the same time); Figures 4-6 The SF9 expressed E2-UGT73CU3 protein, mass spectrometry detects that the reaction does not proceed (no product molecular weight is detected), and the protein has no activity. E. coli E. coli The SF9 expressed E2-GmSGT2 protein is amplified by the system in Table 2, the obtained product is purified by liquid phase preparation, and the purity and yield of the product are detected after freeze-drying. It can be seen that the purity detected by HPLC is 97%, and the yield of the product of the amplification reaction is 81%. At the same time, we detect the structure of the product by nuclear magnetic resonance, and the results are shown in Table 4.
[0062] E. coli The specific raw materials in the present application are all existing substances, which can be directly purchased from the market. Figure 7 Figure 8 The above is only a preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
[0063] The specific raw materials in the present application are all existing substances, which can be directly purchased from the market.
[0064] The above is only a preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A process for the preparation of a QS21 intermediate, characterised in that, The method comprises the following steps: S1, mixing QA-betulinic acid, UDP-D-GlcA and E1-CSLM2 enzyme to react and purifying to obtain QA-GlcA intermediate; S2, mixing QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme to react and purifying to obtain QS21 intermediate QA-GlcA-Gal; In step S1, the E1-CSLM2 enzyme is obtained by insect sf9 expression.
2. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S1, the mixed reaction further comprises pH 7.5 Tris-HCl, MgCl2 and DMSO.
3. A process for the preparation of a QS21 intermediate according to claim 2, characterised in that, In step S1, after mixing, the final concentration of QA-betulinic acid is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.
4. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S1, the temperature of the mixed reaction is 30℃.
5. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S2, the E2-GmSGT2 enzyme is obtained by E. coli expression.
6. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S2, the mixed reaction further comprises pH 7.5 Tris-HCl and MgCl2.
7. A process for the preparation of a QS21 intermediate according to claim 6, characterised in that, In step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.
8. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, The temperature of the mixed reaction is 30℃.